
Full text loading...
Category: Applied and Industrial Microbiology
Molecular Biology of the Model Piezophile, Shewanella violacea DSS12, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555815646/9781555814236_Chap17-1.gif /docserver/preview/fulltext/10.1128/9781555815646/9781555814236_Chap17-2.gifAbstract:
This chapter focuses on the molecular characteristics of pressure adaptation in Shewanella violacea DSS12 and recent advances in developing genetics and utilizing genomics. Sigma 54 plays an important role in pressure-regulated transcription in S. violacea, although it should be noted that the expression of sigma 54 is not itself regulated by pressure. Any of these trans-acting factors (sigma 54, NtrC, or NtrB) could play an important role in pressure-regulated transcription in this piezophilic bacterium. Downstream from the pressure-regulated operon described is an open reading frame (ORF) homologous to the cydD gene of E. coli. Another aspect of transcription in piezophiles is the stability of the quaternary structure of RNA polymerase. It is likely that the S. violacea sigma subunit stabilizes the core enzyme through alteration of the quaternary structure of RNA polymerase, resulting in piezotolerance. One possible explanation for the low conjugation frequency is that the optimal mating temperature for DSS12R is 20°C, which is much lower than the optimal temperature for E. coli. This study provided the first demonstration of gene transfer in the piezophilic bacterium DSS12. The study of the mechanisms for adaptation to high-pressure environments, including gene regulatory systems, may now also proceed in vivo using genetic approaches in this piezophile. Therefore, genomic analysis of marine extremophiles may lead to the discovery of new functions for genes.
Full text loading...
Diagrammatic representation of the pressure-regulated genes in S. violacea strain DSS12. (A) Pressure-regulated operon; (B) glutamine synthetase operon. Bold numbers (#2 in panel A and #1 in panel B) show the transcription start sites controlled by the sigma 54 factor.
Autophosphorylation of the S. violacea NtrB protein, trans-phosphorylation of NtrB-P to the NtrC protein in vitro, and Western blot analysis of expression of the NtrC under different pressure conditions. (A) Autophosphorylation of NtrB incubated in the presence of [γ-32P]ATP at several temperature conditions. Lane 1, 0°C; lane 2, 10°C; lane 3, 24°C; lane 4, 37°C. (B) trans-Phosphorylation to NtrC incubated with the phosphorylated NtrB-P at 10°C for 1 min. Lane 5, phosphorylated NtrB; lane 6, phosphorylated NtrC. (C) Lysates prepared from cells cultured at 0.1 or 50 MPa were fractionated by SDS–10% PAGE and then blotted onto a polyvinylidene fluoride membrane. The membrane was treated with antiserum against NtrC.
Model for pressure regulation of gene expression in the piezophilic S. violacea strain DSS12.
Reduced minus oxidized difference spectra of membrane fractions from S. violacea. Each fraction was obtained from the cells grown under a pressure of 0.1 MPa with shaking (A), grown under a pressure of 0.1 MPa under microaerobic conditions (B), and grown under a pressure of 50 MPa with microaerobic conditions (C). An absorption peak at 629 nm and a trough at 649 nm are specifically detected in the membrane fraction of cells grown under a pressure of 50 MPa. These spectral properties are typical of d-type cytochromes ( 16 ).
Diagram of the HPEA. (A) High-pressure electrophoresis chamber for HPEA.(a) Connection to the power supply (anode); (b) connection to the power supply (cathode); (c) buffers; (d) silicone oil KF-96-1.5CS; (e) glass microcapillary tube; (f) O-ring to partition a space into the upper and lower spaces; (g) connection to a high-pressure pump. (B) Photograph of the HPEA. 1, high-pressure electrophoresis chamber; 2, high-pressure hand pump; 3, pressure gauge.
Effects of high hydrostatic pressure on subunit association in RNA polymerase in E. coli (A), S. violacea (B), and S. violacea without sigma factor (C). Native PAGE was performed at 0.1 MPa (upper panels) or 140 MPa (lower panels), followed by SDS-PAGE at 0.1 MPa. Proteins were visualized by silver staining. Each subunit of RNA polymerase is shown by an open circle. M, molecular mass markers; 1st dim, one-dimensional separation; 2nd dim, two-dimensional separation.
Images of 1% agarose electrophoresis of plasmid extracted from DSS12R transconjugants.
Annotation of ORFs in the S. violacea genome. The total genome size is 4.9 Mbp, and approximately 4,600 ORFs are predicted.
Composition of cytochromes in S. violacea
Comparison of S. violacea DSS12 conjugation efficiencies with different plasmids a